Obesity and diabetes have reached global epidemic status and affect nearly 1 billion and 250 million people, respectively, worldwide. Recently, the peptide hormone, ghrelin, was discovered as a potent stimulator of growth hormone secretion and the only circulatory hormone known to potently enhance feeding and weight gain and to regulate energy homeostasis. Thus, ghrelin has been implicated as a lead anti-obesity therapeutic target. Interestingly, ghrelin contains a novel acylated structure, namely an n-octanoyl post-translational modification, and its biological functions are solely dependent on this modification. Importantly, ghrelin is the only known peptide to contain this novel acylated structure indicating that therapies geared toward blocking the enzyme-mediated addition of this modification could be specific for modulating the activity of ghrelin. The enzyme responsible for this post-translational modification was recently discovered and named ghrelin O-acyltransferase, or GOAT. Murine genetic knockout models of GOAT function have indicated that contrary to previous hypotheses regarding ghrelin's function, the ghrelin-GOAT system seems to be primarily involved in nutrient sensing and maintenance of blood glucose levels. In fact, several ghrelin knockout studies have shown that ablation of this peptide improves diabetic and not obese phenotypes as anticipated. To better dissect the overall impact of the ghrelin-GOAT system on metabolism and energy balance, small molecule chemical probes, which provide temporal control of target protein function, are needed. However, current assays that can be used to screen for modulators of acyltransferase activity are limiting. Thus, in order to identify chemical probes for GOAT, a new approach toward acyltransferase assay design must be taken. Here, we propose to use our recently described fluorescence-based GOAT assay to screen small molecule modulators of GOAT. Using these scaffolds, we hope to answer some of the fundamental questions regarding the role of the ghrelin-GOAT system in the diabetic phenotype, namely in insulin secretion from pancreatic beta cells, and establish these scaffolds as chemical probes for GOAT. Toward the goal of identifying chemical probes for GOAT and providing high-throughput validation of our GOAT assay, our first aim will be to screen for modulators of GOAT activity. Following the identification of compounds that show activity in this initial screen, the compounds will be characterized for in vitro activity, false positives and toxicity. All confirmed hits will then be subjected to confirmatory cellular-based assays that monitor ghrelin production from beta cells and pituitary cells. Using medicinal chemistry, we will optimize all aspects of potency, both in vitro and in cells. In an attempt to examine the impact of the ghrelin-GOAT system in insulin secretion, our second aim will focus on using GOAT-targeted small molecule modulators to investigate the role that these biomolecules play in insulin secretion from pancreatic beta cells. From this aim, we hope to provide more definitive conclusions regarding the relationship between ghrelin and insulin and establish the identified small molecule modulators as effective chemical probes for GOAT.